An atypical type II topoisomerase from Mycobacterium smegmatis with positive supercoiling activity
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Summary Topoisomerases are essential ubiquitous enzymes, falling into two distinct classes. A number of eubacteria including Escherichia coli , typically contain four topoisomerases, two type I topoisomerases and two type II topoisomerases viz. DNA gyrase and topoisomerase IV. In contrast several other bacterial genomes including mycobacteria, encode for one type I topoisomerase and a DNA gyrase. Here we describe a new type II topoisomerase from Mycobacterium smegmatis which is different from DNA gyrase or topoisomerase IV in its characteristics and origin. The topoisomerase is distinct with respect to domain organization, properties and drug sensitivity. The enzyme catalyses relaxation of negatively supercoiled DNA in an ATP‐dependent manner and also introduces positive supercoils to both relaxed and negatively supercoiled substrates. The genes for this additional topoisomerase are not found in other sequenced mycobacterial genomes and may represent a distant lineage.Keywords:
Mycobacterium smegmatis
Topoisomerase inhibitor
Topoisomerase IV
Topoisomerase IV
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We have identified a topoisomerase activity from Escherichia coli related to DNA gyrase (topoisomerase II): we designate it topoisomerase II'. It was constructed of two subunits, which were purified separately. One is the product of the gyrA (formerly nalA) gene and is identical to subunit A of DNA gyrase. The other is a 50,000-dalton protein, which we have purified to homogeneity and call v. v may be a processed form of the much larger gyrase subunit B or may be derived from a transcript of part of the subunit B structural gene, because preliminary peptide maps of the two subunits are similar. Topoisomerase II' relaxes negatively supercoiled DNA and, uniquely among E. coli topoisomerases, relaxes positive supercoils efficiently. It is the only topoisomerase that can introduce positive supercoils; these are stoichiometric with enzyme molecules. Topoisomerase II' resembles gyrase in its sensitivity to oxolinic acid, the wrapping of DNA in an apparent positive supercoil around the enzyme, and the introduction in an aborted reaction of site-specific double-strand breaks in the DNA with concomitant covalent attachment of protein to both newly created 5' ends. Unlike DNA gyrase, topoisomerase II' has no negative supercoiling activity. Functional chimeric topoisomerases were constructed with the alpha subunit of the Micrococcus luteus gyrase and v or gyrase subunit B from E. coli. We discuss the implications of the dual of the gyrA gene product.
Topoisomerase IV
Novobiocin
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Summary Topoisomerases are essential ubiquitous enzymes, falling into two distinct classes. A number of eubacteria including Escherichia coli , typically contain four topoisomerases, two type I topoisomerases and two type II topoisomerases viz. DNA gyrase and topoisomerase IV. In contrast several other bacterial genomes including mycobacteria, encode for one type I topoisomerase and a DNA gyrase. Here we describe a new type II topoisomerase from Mycobacterium smegmatis which is different from DNA gyrase or topoisomerase IV in its characteristics and origin. The topoisomerase is distinct with respect to domain organization, properties and drug sensitivity. The enzyme catalyses relaxation of negatively supercoiled DNA in an ATP‐dependent manner and also introduces positive supercoils to both relaxed and negatively supercoiled substrates. The genes for this additional topoisomerase are not found in other sequenced mycobacterial genomes and may represent a distant lineage.
Mycobacterium smegmatis
Topoisomerase inhibitor
Topoisomerase IV
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As a means of gaining information on the selectivity of quinolone antibacterial agents, we examined their effect on four topoisomerases, topoisomerases I and II purified from Escherichia coli and calf thymus. The inhibition of supercoiling and relaxation activities was monitored by using the classical gel electrophoresis assay. Eight quinolones were assayed by using the four enzymes. Gyrase was much more sensitive to quinolones than the other topoisomerases which can therefore be inhibited by moderate concentrations of certain quinolones. No good correlation was observed between the activity on gyrase and on the other enzymes, since the ratio varied from 15 to more than 8,500. On the contrary, there was a good correlation between early inhibition of DNA synthesis, inhibition of gyrase, and MICs.
Quinolone
Novobiocin
Topoisomerase IV
Topoisomerase inhibitor
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Antibiotic resistance is a major problem estimated to cost $100 trillion and cause 10 million deaths per year by 2050. Despite novel molecules targeting Gram-positive bacteria, there are no new antibiotics active against Gram-negatives. To prolong use of current drugs, we need to understand mechanisms of resistance to inform prescribing practices and drug discovery. Quinolone resistance is primarily conferred by mutations in the target loci: DNA gyrase (gyrA) and topoisomerase IV. Quinolone resistance arising from gyrA mutations has also been shown to confer a low level of protection against a range of non-quinolone drugs. This thesis investigated the hypotheses that altered supercoiling levels, resulting from gyrA mutations, alter expression of stress response genes and confer a generic protective effect against other antibiotics and chemicals. The effects of equivalent gyrA mutations in Salmonella and E. coli upon supercoiling were analysed. Both GyrA Ser83Phe and GyrA Asp87Gly substitutions resulted in altered topoisomer profiles, although these were different between the species. When exposed to stresses, Salmonella gyrA mutants maintain supercoiling in a relatively fixed manner, providing a degree of antimicrobial protection but possibly limiting flexibility in response to environmental change. Fluorescent reporter assays showed a modest elevation of stress responses in Salmonella GyrA Asp87Gly cells, but highly upregulated stress responses in E. coli GyrA Asp87Gly cells. This correlated with a competitive fitness benefit of E. coli GyrA Asp87Gly cells vs the parent in the presence of low levels of triclosan. The elevated stress responses likely result from supercoiling-induced changes in promoter accessibility, and are probably responsible for the generic protective effect gyrA mutation confers against other chemicals and antibiotics. Non-quinolone antimicrobials can provide a selective pressure that favours gyrA mutants, although this is highly dependent on condition and species.
Quinolone
Topoisomerase IV
Chemostat
SOS response
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Although bacterial gyrase and topoisomerase IV have critical interactions with positively supercoiled DNA, little is known about the actions of these enzymes on overwound substrates. Therefore, the abilities of Bacillus anthracis and Escherichia coli gyrase and topoisomerase IV to relax and cleave positively supercoiled DNA were analyzed. Gyrase removed positive supercoils ∼10-fold more rapidly and more processively than it introduced negative supercoils into relaxed DNA. In time-resolved single-molecule measurements, gyrase relaxed overwound DNA with burst rates of ∼100 supercoils per second (average burst size was 6.2 supercoils). Efficient positive supercoil removal required the GyrA-box, which is necessary for DNA wrapping. Topoisomerase IV also was able to distinguish DNA geometry during strand passage and relaxed positively supercoiled substrates ∼3-fold faster than negatively supercoiled molecules. Gyrase maintained lower levels of cleavage complexes with positively supercoiled (compared with negatively supercoiled) DNA, whereas topoisomerase IV generated similar levels with both substrates. Results indicate that gyrase is better suited than topoisomerase IV to safely remove positive supercoils that accumulate ahead of replication forks. They also suggest that the wrapping mechanism of gyrase may have evolved to promote rapid removal of positive supercoils, rather than induction of negative supercoils.
Topoisomerase IV
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DNA supercoiling is essential for bacterial cell survival. We demonstrated that DNA topoisomerase IV, acting in concert with topoisomerase I and gyrase, makes an important contribution to the steady-state level of supercoiling in Escherichia coli. Following inhibition of gyrase, topoisomerase IV alone relaxed plasmid DNA to a final supercoiling density (ς) of −0.015 at an initial rate of 0.8 links min−1. Topoisomerase I relaxed DNA at a faster rate, 5 links min−1, but only to a ς of −0.05. Inhibition of topoisomerase IV in wild-type cells increased supercoiling to approximately the same level as in a mutant lacking topoisomerase I activity (to ς = −0.08). The role of topoisomerase IV was revealed by two functional assays. Removal of both topoisomerase I and topoisomerase IV caused the DNA to become hyper-negatively supercoiled (ς = −0.09), greatly stimulating transcription from the supercoiling sensitive leu-500promoter and increasing the number of supercoils trapped by λ integrase site-specific recombination.
Topoisomerase IV
Transcription
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Changes in plasmid DNA supercoiling were measured following treatment of Escherichia coli cells, carrying topoisomerase mutations, with the quinolone ciprofloxacin. In quinolone-susceptible cells (top+ gyr+) as well as in topA mutants and in gyrB mutants, plasmid DNA was relaxed after the addition of ciprofloxacin. In cells partially resistant to quinolones, low ciprofloxacin levels led to an increase in negative superhelicity of plasmid DNA, whereas at higher ciprofloxacin concentrations, DNA became relaxed. Cells exhibiting partial resistance to quinolones carried either a gyrA mutation alone or a combination of gyrA and gyrB mutations. Moreover, they showed a reduction in gyrase activity, indicated by the supercoiling of a reporter plasmid. Therefore, we conclude that a low level of quinolone action and a DNA with a lower-than-normal level of superhelicity are the two essential conditions for obtaining a ciprofloxacin-promoted increase in plasmid DNA supercoiling. In contrast, deficiency in topoisomerase I is not required for this effect.
Topoisomerase IV
Quinolone
Sparfloxacin
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Topoisomerase IV
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Abstract To perform double-stranded DNA passage, type II topoisomerases generate a covalent enzyme-cleaved DNA complex (i.e. cleavage complex). Although this complex is a requisite enzyme intermediate, it is also intrinsically dangerous to genomic stability. Consequently, cleavage complexes are the targets for several clinically relevant anticancer and antibacterial drugs. Human topoisomerase IIα and IIβ and bacterial gyrase maintain higher levels of cleavage complexes with negatively supercoiled over positively supercoiled DNA substrates. Conversely, bacterial topoisomerase IV is less able to distinguish DNA supercoil handedness. Despite the importance of supercoil geometry to the activities of type II topoisomerases, the basis for supercoil handedness recognition during DNA cleavage has not been characterized. Based on the results of benchtop and rapid-quench flow kinetics experiments, the forward rate of cleavage is the determining factor of how topoisomerase IIα/IIβ, gyrase and topoisomerase IV distinguish supercoil handedness in the absence or presence of anticancer/antibacterial drugs. In the presence of drugs, this ability can be enhanced by the formation of more stable cleavage complexes with negatively supercoiled DNA. Finally, rates of enzyme-mediated DNA ligation do not contribute to the recognition of DNA supercoil geometry during cleavage. Our results provide greater insight into how type II topoisomerases recognize their DNA substrates.
Cleavage (geology)
Topoisomerase IV
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